SUMMARY The genetic basis of social behavior is complex and involves many genes spread throughout the genome. As a result, it has been difficult to link specific genes to social behavior in humans. Increasing accessibility to genomic resources has now made it feasible to draw such connections in novel animal models. Well-chosen models make possible an experimental, mechanistic strategy that begins at the level of gene variation and works up through layers of biological organization, connecting each level to the one below, to explain the effect of genetic variation on behavioral phenotype. The proposed research will leverage a large body of previous work, conducted by the PI and collaborators, in a natural animal model uniquely suited for connecting genes and social behavior. In the white-throated sparrow (Zonotrichia albicollis), a common North American songbird, an inversion on chromosome 2 (ZAL2m) has significantly altered social strategies. Limited gene flow between the ZAL2 and ZAL2m haplotypes has driven genetic differentiation of the rearranged region. As a direct result, ZAL2m individuals of both sexes employ a life history strategy different from that of their ZAL2 counterparts ? they are more aggressive and less parental. The long-term goal is to use this inversion as a target to show how a small genetic change can have large downstream effects on complex behavioral phenotypes. The PI and collaborators have already identified a limited number of genes located inside the inversion that meet the following criteria: (1) expression differs between birds with and without the inversion, (2) the genes show high allele-specific expression in heterozygotes, and (3) expression is tightly correlated with vocal aggression. These genes include a neuropeptide previously implicated in aggression, a serotonin receptor, and a glutamate receptor. The objective of the proposed renewal application is to systematically evaluate the causal effects of variation in these genes on aggressive phenotypes, working up through multiple levels of biological organization from genotype to phenotype. First, the team will precisely identify the genetic variation responsible for differential expression of these genes. Second, they will use bottom-up and targeted proteomics to show the impact of genetic differentiation on the expression of protein isoforms in the brain. Finally, they will manipulate production of the relevant proteins to test their roles in aggressive behavioral phenotypes. A major strength of the research strategy is that the team will combine discovery-based, genome-wide approaches with hypothesis-driven, targeted approaches, using the former to inform the latter. The project is innovative because it takes advantage of a unique, powerful model organism that can be studied in its natural habitat. The significance of this work is that it will move beyond genotype-phenotype associations to test for causal connections between gene variants and social behavior, at multiple levels of biological organization, in a vertebrate. Using this strategy, the investigators will advance understanding of not only which genes contribute to behavioral phenotypes, but how they do so.